T lymphocytes are responsible primarily for protection against intracellular pathogens and malignancies. Individuals who are grossly deficient in T-cell immunity frequently succumb to overwhelming infections by organisms such as cytomegalovirus, Pneumocystis carinii, Candida, and other apparently "opportunistic" pathogens, including bacteria, viruses and fungi. These individuals may also succumb to malignancies such as B cell lymphomas, indicating the importance of T cell immunity in the suppression or elimination of certain tumors. Immunosuppression can result from a variety of causes, including viral infections (for example, with the RIV virus), as a result of chemical therapy, and malignancies (particularly of types that affect the hematopoietic system).
All mature T lymphocytes express the CD3 cell surface molecule, but consist of two basic subtypes based on their mutually exclusive expression of cell surface molecules CD4 and CD8. The functional distinction between CD4+ and CD8+ T cells is based on the ability of CD4+ cells to recognize antigen presented in association with class II MHC molecules, and CD8+ cells to recognize antigen presented in association with class I MHC molecules. CD8+ cells are involved in "effector" functions in immune responses, such as direct cytotoxic destruction of target cells bearing foreign antigens, and represent an important mechanism for resistance to viral infections and tumors. The CD8+ cells that mediate this lytic function are designated cytotoxic T lymphocytes (CTLs). CD4+ T cells are generally involved in "helper" functions in immune responses and secrete cytokine molecules, in particular IL-2, upon which the cytotoxic CD8+ T cells are dependent. CD4+ T cells are often referred to as T helper (TH) cells. Although most CTL are of the CD8+ phenotype, some CTL of the CD4+ phenotype have been described. Generally, individual CTLs (whether CD8+ or CD4+) are antigen-specific.
Classes of lymphocytes, for example CTLs, are dependent on helper T (TH) cell-derived cytokines, such as IL-2, for growth and proliferation in response to foreign antigens. (Zinkernagel and Doherty, Adv. Immunol. 27:51, 1979; Male et al., Advanced Immunology, Chap. 7, Gower Publ., London, 1987; Jacobson et al., J. Immunol. 133:754, 1984). IL-2, for example, is a potent mitogen for cytotoxic T lymphocytes (Gillis and Smith, Nature 268:154, 1977), and the combination of antigen and IL-2 cause proliferation of primary CD8+ T cells in vitro. The importance of IL-2 for the growth and maintenance of the CD8+ CTL in vivo has been documented in models of adoptive immunotherapy in which the therapeutic efficacy of transferred anti-retroviral CD8+ cells is enhanced on subsequent administration of IL-2 (Cheever et al., J. Exp. Med. 155:968, 1982; Reddehase et al., J. Virol. 61:3102, 1987). IL-4 and IL-7 are also capable of stimulating the proliferation of mature CD8+ CTL (Alderson et al., J. Exp. Med. 172:577, 1990).
Considerable research has been focused on the use of T cells in treating malignant tumors and viral infections. Cytotoxic T cells specific for a particular type of tumor can be isolated and administered to a patient having a tumor, with the effect that the CTLs ameliorate the tumor. It has been demonstrated, for example, that tumor-specific T cells can not only be generated to experimental tumors in mice, but also that T cells with apparent tumor specificity can be isolated from human tumors. Such human tumor infiltrating lymphocytes (TILs) have been expanded in vitro and used to treat cancer patients, generating significant enthusiasm for human adoptive immunotherapy with tumor-specific T cells (Rosenberg et al., N. Engl. J. Med. 319:1767, 1988).
Similar studies using cytotoxic T cells specific for viral antigens have also been conducted in animal models. Human HIV-specific CTL of both the CD8+ (Walker et al., Nature 328:345, 1987; Plata et al., Nature 328:348, 1987) and CD4+ (Siliciano et al., Cell 54:561, 1988) phenotype have been isolated and characterized. HIV-specific CD8+ CTL are classical CTL in that their proliferative and cytotoxic responses are antigen-specific and MHC-restricted (Walker et al., supra; Chenciner et al., Eur. J. Immuno. 19:1537, 1989; Walker et al., Proc. Natl. Acad. Sci. USA 86:9514, 1989), in common with the numerous mouse and human CTL clones which have been characterized which are specific for viral, tumor or allospecific antigens.
The adoptive transfer of antigen (Ag)-specific T cells to establish immunity appears to be an effective therapy for some viral infections and tumors in the mouse animal model system. (For a review, See P. D. Greenberg, in Advances in Immunology F. Dixon Ed. Academic Press, Inc. Orlando Fla. (1991), pp. 280-355.) However, a successful outcome of an adoptive transfer method is dependent upon many factors, including the longevity of the transferred clones and the lack of toxicity to the host of the transferred cells. Although many antigen-specific T cell clones have been isolated, the use of tumor-specific T cell clones generated in vitro has been shown to have definite limitations in tumor therapy. It has been demonstrated in several therapeutic models that the efficacy of cytolytic CD8+ T cells is limited by a dependency on exogenous IL-2 (produced by TH cells), a finding that has been substantiated in human adoptive therapy trials in which administration of exogenous IL-2 appears essential for optimal therapeutic efficacy (Rosenberg et al., N. Engl. J. Med. 319:1767;, 1988; Klarnet et al., in Role of Interleukin-2 Activated Killer Cells in Cancer, Lutzova and Herberman (eds.), CRC Press, Florida, Chap. 14, pp. 199-218, 1990). Thus, while in vitro T cell cloning techniques provide a means to generate large numbers of T cells with demonstrable tumor or viral specificity, the full potential of using such antigen-specific T cells in therapy appears to be limited by their dependency on TH cells.
In some limited instances the problem of TH dependency may be circumvented by using a particular class of cells known to function independent of TH cells. These cells are known as helper-independent cytolytic CD8+ cells (HITc) (Klarnet et al., J. Immunol. 142:2187, 1989) and have been identified in populations of primary (i.e., freshly isolated from in vivo sources) CD8+ CTL (Sprent and Schaefer, J. Exp. Med. 162:21068, 1985; Andrus et al., J. Exp. Med. 159:647, 1984). HITc cells produce sufficient IL-2 to grow independently of CD4+ cells and the cytokines they produce. HITc cells are also known to express plasma membrane IL-1 receptors (IL-1R) and require IL-1 for their IL-2-independent proliferation (Klarnet et al., 1989, supra). This is in contrast to conventional CD8+ CTL which do not express detectable IL-1R on their surface (Lowenthal and MacDonald, 1987). HITc cells have been generated which are specific for a range of antigens, including tumor, viral and alloantigens (von Boehmer et al., 1. Immunol. 133:59, 1984; Klarnet et al., J. Immunol. 138:4012, 1987; and Andrus et al., J. Exp. Med. 149:647, 1984; Mizouchi et al., J. Immunol. 142:270, 1989). HITc specific for a retrovirally transformed tumor have been shown to eradicate the tumor cells and persist long-term in vivo following their engraftment (Klarnet et al., 1989, supra). Unfortunately, HITc cells having specificity for many important antigens, such as HIV antigens or tumor antigens, have not yet been isolated.
In order to realize the full potential of antigen-specific T cells in therapy, it is necessary to develop a more complete repertoire of CTLs that have a lessened dependency on TH cells. One approach has been the introduction of a recombinant vector that expresses a cytokine receptor, for example the IL-1 receptor, into TH-dependent CTL, resulting in the conversion to cells with a lessened requirement for the TH cells and/or their stimulatory factors. (PCT/US91/06921, WO 92/05794, published Apr. 16, 1992). The present invention, described below, presents a different approach to the production of lymphocytes with a lessened dependency on TH cells and/or the stimulatory factors they produce.